Motile cilia, also called flagella, are found across a broad range of species; some cilia propel prokaryotes and eukaryotic cells like sperm, while cilia on epithelial surfaces create complex fluid patterns e.g., in the brain or lung. For sperm, the picture has emerged that the flagellum is not only a motor but also a sensor that detects stimuli from the environment, computing the beat pattern according to the sensory input. Thereby, the flagellum navigates sperm through the complex environment in the female genital tract. However, we know very little about how environmental signals change the flagellar beat and, thereby, the swimming behavior of sperm. It has been proposed that distinct signaling domains in the flagellum control the flagellar beat. However, a detailed analysis has been mainly hampered by the fact that current comprehensive analysis approaches rely on complex microscopy and analysis systems. Thus, knowledge on sperm signaling regulating the flagellar beat is based on custom quantification approaches that are limited to only a few aspects of the beat pattern, do not resolve the kinetics of the entire flagellum, rely on manual, qualitative descriptions, and are only a little comparable among each other. Here, we present SpermQ, a ready-to-use and comprehensive analysis software to quantify sperm motility. SpermQ provides a detailed quantification of the flagellar beat based on common time-lapse images acquired by dark-field or epi-fluorescence microscopy, making SpermQ widely applicable. We envision SpermQ becoming a standard tool in flagellar and motile cilia research that allows to readily link studies on individual signaling components in sperm and distinct flagellar beat patterns.

Many biological processes happen on a nano- to millimeter scale and within milliseconds. Established methods such as confocal microscopy are suitable for precise 3D recordings but lack the temporal or spatial resolution to resolve fast 3D processes and require labeled samples. Multifocal imaging (MFI) allows high-speed 3D imaging but is limited by the compromise between high spatial resolution and large field-of-view (FOV), and the requirement for bright fluorescent labels. Here, we provide an open-source 3D reconstruction algorithm for multi-focal images that allows using MFI for fast, precise, label-free tracking spherical and filamentous structures in a large FOV and across a high depth. We characterize fluid flow and flagellar beating of human and sea urchin sperm with a z-precision of 0.15 µm, in a volume of 240 × 260 × 21 µm, and at high speed (500 Hz). The sampling volume allowed to follow sperm trajectories while simultaneously recording their flagellar beat. Our MFI concept is cost-effective, can be easily implemented, and does not rely on object labeling, which renders it broadly applicable.

Shuttling RNA-binding proteins coordinate nuclear and cytoplasmic steps of gene expression. The SR family proteins regulate RNA splicing in the nucleus and a subset of them, including SRSF1, shuttles between the nucleus and cytoplasm affecting post-splicing processes. However, the physiological significance of this remains unclear. Here, we used genome editing to knock-in a nuclear retention signal (NRS) in Srsf1 to create a mouse model harboring an SRSF1 protein that is retained exclusively in the nucleus. Srsf1NRS/NRS mutants displayed small body size, hydrocephalus, and immotile sperm, all traits associated with ciliary defects. We observed reduced translation of a subset of mRNAs and decreased abundance of proteins involved in multiciliogenesis, with disruption of ciliary ultrastructure and motility in cells and tissues derived from this mouse model. These results demonstrate that SRSF1 shuttling is used to reprogram gene expression networks in the context of high cellular demands, as observed here, during motile ciliogenesis.

The different adipose tissues (ATs) can be distinguished according to their function. For example, white AT stores energy in form of lipids, whereas brown AT dissipates energy in the form of heat. These functional differences are represented in the respective adipocyte morphology; whereas white adipocytes contain large, unilocular lipid droplets, brown adipocytes contain smaller, multilocular lipid droplets. However, an automated, image analysis pipeline to comprehensively analyze adipocytes in vitro in cell culture as well as ex vivo in tissue sections is missing. We here present AdipoQ, an open-source software implemented as ImageJ plugins that allows us to analyze adipocytes in tissue sections and in vitro after histological and/or immunofluorescent labeling. AdipoQ is compatible with different imaging modalities and staining methods, allows batch processing of large datasets and simple post-hoc analysis, provides a broad band of parameters, and allows combining multiple fluorescent readouts. Therefore AdipoQ is of immediate use not only for basic research but also for clinical diagnosis.

Motile cilia are miniature, propeller-like extensions, emanating from many cell types across the body. Their coordinated beating generates a directional fluid flow, which is essential for various biological processes, from respiration to reproduction. In the nervous system, ependymal cells extend their motile cilia into the brain ventricles and contribute to cerebrospinal fluid (CSF) flow. Although motile cilia are not the only contributors to CSF flow, their functioning is crucial, as patients with motile cilia defects develop clinical features, like hydrocephalus and scoliosis. CSF flow was suggested to primarily deliver nutrients and remove waste, but recent studies emphasized its role in brain development and function. Nevertheless, it remains poorly understood how ciliary beating generates and organizes CSF flow to fulfill these roles. Here, we study motile cilia and CSF flow in the brain ventricles of larval zebrafish. We identified that different populations of motile ciliated cells are spatially organized and generate a directional CSF flow powered by ciliary beating. Our investigations revealed that CSF flow is confined within individual ventricular cavities, with little exchange of fluid between ventricles, despite a pulsatile CSF displacement caused by the heartbeat. Interestingly, our results showed that the ventricular boundaries supporting this compartmentalized CSF flow are abolished during bodily movement, highlighting that multiple physiological processes regulate the hydrodynamics of CSF flow. Finally, we showed that perturbing cilia reduces hydrodynamic coupling between the brain ventricles and disrupts ventricular development. We propose that motile-cilia-generated flow is crucial in regulating the distribution of CSF within and across brain ventricles.

The flagellum is essential for sperm motility and fertilization in vivo. The axoneme is the main component of the flagella, extending through its entire length. An axoneme is comprised of two central microtubules surrounded by nine doublets, the nexin-dynein regulatory complex, radial spokes, and dynein arms. Failure to properly assemble components of the axoneme in a sperm flagellum, leads to fertility alterations. To understand this process in detail, we have defined the function of an uncharacterized gene, Cfap97 domain containing 1 (Cfap97d1). This gene is evolutionarily conserved in mammals and multiple other species, including Chlamydomonas. We have used two independently generated Cfap97d1 knockout mouse models to study the gene function in vivo. Cfap97d1 is exclusively expressed in testes starting from post-natal day 20 and continuing throughout adulthood. Deletion of the Cfap97d1 gene in both mouse models leads to sperm motility defects (asthenozoospermia) and male subfertility. In vitro fertilization (IVF) of cumulus-intact oocytes with Cfap97d1 deficient sperm yielded few embryos whereas IVF with zona pellucida-free oocytes resulted in embryo numbers comparable to that of the control. Knockout spermatozoa showed abnormal motility characterized by frequent stalling in the anti-hook position. Uniquely, Cfap97d1 loss caused a phenotype associated with axonemal doublet heterogeneity linked with frequent loss of the fourth doublet in the sperm stored in the epididymis. This study demonstrates that Cfap97d1 is required for sperm flagellum ultra-structure maintenance, thereby playing a critical role in sperm function and male fertility in mice.

Sialic acid-binding Ig-like lectin (Siglec) receptors are linked to neurodegenerative processes, but the role of sialic acids in physiological aging is still not fully understood. We investigated the impact of reduced sialylation in the brain of mice heterozygous for the enzyme glucosamine-2-epimerase/N-acetylmannosamine kinase (GNE+/-) that is essential for sialic acid biosynthesis. We demonstrate that GNE+/- mice have hyposialylation in different brain regions, less synapses in the hippocampus and reduced microglial arborization already at 6 months followed by increased loss of neurons at 12 months. A transcriptomic analysis revealed no pro-inflammatory changes indicating an innate homeostatic immune process leading to the removal of synapses and neurons in GNE+/- mice during aging. Crossbreeding with complement C3-deficient mice rescued the earlier onset of neuronal and synaptic loss as well as the changes in microglial arborization. Thus, sialic acids of the glycocalyx contribute to brain homeostasis and act as a recognition system for the innate immune system in the brain.

Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes, have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/yCylc2+/-, and Cylc1-/yCylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.

Astrocytic hyperactivity is an important contributor to neuronal-glial network dysfunction in Alzheimer's disease (AD). We have previously shown that astrocyte hyperactivity is mediated by signaling through the P2Y1 purinoreceptor (P2Y1R) pathway. Using the APPPS1 mouse model of AD, we here find that chronic intracerebroventricular infusion of P2Y1R inhibitors normalizes astroglial and neuronal network dysfunction, as measured by in vivo two-photon microscopy, augments structural synaptic integrity, and preserves hippocampal long-term potentiation. These effects occur independently from β-amyloid metabolism or plaque burden but are associated with a higher morphological complexity of periplaque reactive astrocytes, as well as reduced dystrophic neurite burden and greater plaque compaction. Importantly, APPPS1 mice chronically treated with P2Y1R antagonists, as well as APPPS1 mice carrying an astrocyte-specific genetic deletion (Ip3r2-/-) of signaling pathways downstream of P2Y1R activation, are protected from the decline of spatial learning and memory. In summary, our study establishes the restoration of network homoeostasis by P2Y1R inhibition as a novel treatment target in AD.

Microglia are the primary resident innate immune cells of the CNS. They possess branched, motile cell processes that are important for their cellular functions. To study the pathways that control microglial morphology and motility under physiological and disease conditions, it is necessary to quantify microglial morphology and motility precisely and reliably. Several image analysis approaches are available for the quantification of microglial morphology and motility. However, they are either not automated, not freely accessible, and/or limited in the number of morphology and motility parameters that can be assessed. Thus, we have developed MotiQ, an open-source, freely accessible software for automated quantification of microglial motility and morphology. MotiQ allows quantification of a diverse set of cellular motility and morphology parameters, including the parameters that have become the gold standard in the microglia field. We demonstrate that MotiQ can be applied to in vivo, ex vivo, and in vitro data from confocal, epifluorescence, or two-photon microscopy, and we compare its results to other analysis approaches. We suggest MotiQ as a versatile and customizable tool to study microglia.

The primary cilium constitutes an organelle that orchestrates signal transduction independently from the cell body. Dysregulation of this intricate molecular architecture leads to severe human diseases, commonly referred to as ciliopathies. However, the molecular underpinnings how ciliary signaling orchestrates a specific cellular output remain elusive. By combining spatially resolved optogenetics with RNA sequencing and imaging, we reveal a novel cAMP signalosome that is functionally distinct from the cytoplasm. We identify the genes and pathways targeted by the ciliary cAMP signalosome and shed light on the underlying mechanisms and downstream signaling. We reveal that chronic stimulation of the ciliary cAMP signalosome transforms kidney epithelia from tubules into cysts. Counteracting this chronic cAMP elevation in the cilium by small molecules targeting activation of phosphodiesterase-4 long isoforms inhibits cyst growth. Thereby, we identify a novel concept of how the primary cilium controls cellular functions and maintains tissue integrity in a specific and spatially distinct manner and reveal novel molecular components that might be involved in the development of one of the most common genetic diseases, polycystic kidney disease.

Compartmentalization of cellular signaling forms the molecular basis of cellular behavior. The primary cilium constitutes a subcellular compartment that orchestrates signal transduction independent from the cell body. Ciliary dysfunction causes severe diseases, termed ciliopathies. Analyzing ciliary signaling has been challenging due to the lack of tools to investigate ciliary signaling. Here, we describe a nanobody-based targeting approach for optogenetic tools in mammalian cells and in vivo in zebrafish to specifically analyze ciliary signaling and function. Thereby, we overcome the loss of protein function observed after fusion to ciliary targeting sequences. We functionally localized modifiers of cAMP signaling, the photo-activated adenylyl cyclase bPAC and the light-activated phosphodiesterase LAPD, and the cAMP biosensor mlCNBD-FRET to the cilium. Using this approach, we studied the contribution of spatial cAMP signaling in controlling cilia length. Combining optogenetics with nanobody-based targeting will pave the way to the molecular understanding of ciliary function in health and disease.

The infertility of many couples rests on an enigmatic dysfunction of the man's sperm. To gain insight into the underlying pathomechanisms, we assessed the function of the sperm-specific multisubunit CatSper-channel complex in the sperm of almost 2,300 men undergoing a fertility workup, using a simple motility-based test. We identified a group of men with normal semen parameters but defective CatSper function. These men or couples failed to conceive naturally and upon medically assisted reproduction via intrauterine insemination and in vitro fertilization. Intracytoplasmic sperm injection (ICSI) was, ultimately, required to conceive a child. We revealed that the defective CatSper function was caused by variations in CATSPER genes. Moreover, we unveiled that CatSper-deficient human sperm were unable to undergo hyperactive motility and, therefore, failed to penetrate the egg coat. Thus, our study provides the experimental evidence that sperm hyperactivation is required for human fertilization, explaining the infertility of CatSper-deficient men and the need of ICSI for medically assisted reproduction. Finally, our study also revealed that defective CatSper function and ensuing failure to hyperactivate represents the most common cause of unexplained male infertility known thus far and that this sperm channelopathy can readily be diagnosed, enabling future evidence-based treatment of affected couples.

Accumulating data support the role of tau pathology in cognitive decline in ageing and Alzheimer's disease, but underlying mechanisms remain ill-defined. Interestingly, ageing and Alzheimer's disease have been associated with an abnormal upregulation of adenosine A2A receptor (A2AR), a fine tuner of synaptic plasticity. However, the link between A2AR signalling and tau pathology has remained largely unexplored. In the present study, we report for the first time a significant upregulation of A2AR in patients suffering from frontotemporal lobar degeneration with the MAPT P301L mutation. To model these alterations, we induced neuronal A2AR upregulation in a tauopathy mouse model (THY-Tau22) using a new conditional strain allowing forebrain overexpression of the receptor. We found that neuronal A2AR upregulation increases tau hyperphosphorylation, potentiating the onset of tau-induced memory deficits. This detrimental effect was linked to a singular microglial signature as revealed by RNA sequencing analysis. In particular, we found that A2AR overexpression in THY-Tau22 mice led to the hippocampal upregulation of C1q complement protein-also observed in patients with frontotemporal lobar degeneration-and correlated with the loss of glutamatergic synapses, likely underlying the observed memory deficits. These data reveal a key impact of overactive neuronal A2AR in the onset of synaptic loss in tauopathies, paving the way for new therapeutic approaches.

Mammalian oocytes are enveloped by the zona pellucida (ZP), an extracellular matrix of glycoproteins. In sperm, stimulation with ZP proteins evokes a rapid Ca2+ influx via the sperm-specific, pH-sensitive Ca2+ channel CatSper. However, the physiological role and molecular mechanisms underlying ZP-dependent activation of CatSper are unknown. Here, we delineate the sequence of ZP-signaling events in mouse sperm. We show that ZP proteins evoke a rapid intracellular pH i increase that rests predominantly on Na+/H+ exchange by NHA1 and requires cAMP synthesis by the soluble adenylyl cyclase sAC as well as a sufficiently negative membrane potential set by the spem-specific K+ channel Slo3. The alkaline-activated CatSper channel translates the ZP-induced pH i increase into a Ca2+ response. Our findings reveal the molecular components underlying ZP action on mouse sperm, opening up new avenues for understanding the basic principles of sperm function and, thereby, mammalian fertilization.

Psychosocial stress is one of the main environmental factors contributing to the development of psychiatric disorders. In humans and rodents, chronic stress is associated with elevated inflammatory responses, indicated by increased numbers of circulating myeloid cells and activation of microglia, the brain-resident immune cells. The endocannabinoid system (ECS) regulates neuronal and endocrine stress responses via the cannabinoid receptor 1 (CB1). CB1-deficient mice (Cnr1-/-) are highly sensitive to stress, but if this involves altered inflammatory responses is not known. To test this, we exposed Cnr1+/+ and Cnr1-/- mice to chronic social defeat stress (CSDS). Cnr1-/- mice were extremely sensitive to a standard protocol of CSDS, indicated by an increased mortality rate. Therefore, a mild CSDS protocol was established, which still induced a behavioural phenotype in susceptible Cnr1-/- mice. These mice also showed altered glucocorticoid levels after mild CSDS, suggesting dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mild CSDS induced weak myelopoiesis in the periphery, but no recruitment of myeloid cells to the brain. In contrast, mild CSDS altered microglial activation marker expression and morphology in Cnr1-/- mice. These microglial changes correlated with the severity of the behavioural phenotype. Furthermore, microglia of Cnr1-/- mice showed increased expression of Fkbp5, an important regulator of glucocorticoid signalling. Overall, the results confirm that CB1 signalling protects the organism from the physical and emotional harm of social stress and implicate endocannabinoid-mediated modulation of microglia in the development of stress-related pathologies.